Compendium of Methods to Uncover RNA-Protein Interactions In Vivo

Abstract

Control of gene expression is critical in shaping the pro-and eukaryotic organisms' genotype and phenotype. The gene expression regulatory pathways solely rely on protein-protein and protein-nucleic acid interactions, which determine the fate of the nucleic acids. RNA-protein interactions play a significant role in co- and post-transcriptional regulation to control gene expression. RNA-binding proteins (RBPs) are a diverse group of macromolecules that bind to RNA and play an essential role in RNA biology by regulating pre-mRNA processing, maturation, nuclear transport, stability, and translation. Hence, the studies aimed at investigating RNA-protein interactions are essential to advance our knowledge in gene expression patterns associated with health and disease. Here we discuss the long-established and current technologies that are widely used to study RNA-protein interactions in vivo. We also present the advantages and disadvantages of each method discussed in the review.

Keywords: RNA; RNA–protein interactions; gene expression and post-transcriptional gene regulation; ribonucleoproteins.

Figure 1

Observed conformational changes during RNA–protein interactions. Protein-induced RNA folding (A), RNA-induced protein folding (B), and co-induced folding (C) [3].

Figure 2

Comparison between the crosslinking and immunoprecipitation (CLIP) protocols.

Figure 3

Three-hybrid system to detect and analyze RNA–protein interactions. A three-hybrid system detects RNA–protein interactions. The hybrid RNA interacts with two separate proteins with RNA binding domains that independently interact with proteins containing DNA binding domain and an activation domain, respectively. Once this tripartite complex is formed successfully at the promoter, the reporter gene is activated, serving as a detection method [29,48].

Figure 4

TriFC method in living cells. The TriFC helps detect RNA-protein interaction in living cells. (A) Two complementary portions of the Venus fluorescent protein are attached to a reporter mRNA by the MS2 coat protein and an RNA-binding protein, respectively. (B) If the RNA-binding protein finds a preferred sequence within the reporter mRNA and binds there, the two portions of Venus protein will be brought into proximity to form a fluorescent product [55].

Figure 5

Schematic representation of the strategy utilized to visualize RNA–protein interactions in cells. Fluorescence resonance energy transfer (FRET) signals between the RNA-binding protein (RBP) (protein of interest) bound to the ECFP and MS2-EYFP pairs are detected by the acceptor photobleaching method. This method detects the de-quenched donor fluorophores in the presence of acceptors [62].

Figure 6

Schematic of the PAIR technology. A method using peptide-nucleic-acid-assisted identification of RBPs [63,66]. Peptide nucleic acid (PNA), cell membrane-penetrating peptide (CPP), p-benzoylphenylalanine (BPA).